1. Field of the Invention
This invention relates to bacteriophage-encoded enzymes useful in preventing dental caries and periodontal diseases. More specifically, this invention relates to lysozyme-like enzymes isolated from bacteriophages which are capable of killing cariogenic bacteria and other periodontal disease-causing organisms. The invention also relates to dextranase-like enzymes suitable for dental treatments (i.e., loosening plaque) and other applications where it is desired to remove dextran and other bacterial polysaccharides (i.e., mutan) synthesized from sucrose.
2. Description of the Related Art
Phages have been known to be present in the human mouth for many years (Meyers, C. E. et al (1958) J. Dent. Res. 37:175-178; Natkin, E. (1967) Archs. Oral Biol. 12:669-680; Shimizu, Y. (1968) Odontology 55:583-541) and have been isolated for several genera of oral bacteria, including oral enterococci (Natkin, E. (1967) Archs. Oral Biol. 12:669-680; Smyth, C. J. et al (1987) J. Med. Microbial. 23:45-54), Lactobacillus sp. (Meyers, C. E. et al (1958) J. Dent. Res. 37:175-178; Stetter, K. O. (1977) J. Virol. 24:685-689; Tohyama, K. (1972) Japan. J. Microb. 16:385-395), Veillonella sp. (Shimizu, Y. (1968) Odontology (Japan) 55:583-541; Totsuka, N. (1976) Bull. Tokyo Med. Dent. Univ. 23:261-273), Actinomyces sp. (Bousque, J. L. et al (1988) Ann. Mtg. IADR, J. Dent. Res. 67:394. Abstr. No. 2253; Delisle, A. L. et al (1978) Infect. Immun. 20:303-306; Tylenda, C. A. et al (1985) Infect. Immun. 49:1-6), Streptococcus mutans and S. sobrinus (Armau, E. J. et al (1988) Ann. Mtg. IADR, J. Dent. Res. 67:121 Abstr. No. 69), Actinobacillus actinomycetemcomitans (Preus, H. R. et al (1987) J. Clin. Periodontol. 14: 245-247; Preus, H. R. et al (1987) J. Clin. Periodontol. 14: 605-609; Stevens, R. H. et al (1982) Infect. Immun. 35:343-349), S. sanguis (Parsons, C. L. et al (1972) J. Biol. 9:876-878; Parsons, C. L. et al (1973) J. Bacteriol. 113:1217-1222; Parsons, C. L. et al (1973) J. Bacteriol. 113:1505-1506), Bacteroides buccalis (Tylenda, C. A. et al (1987) Absts. Ann. Mtg., Amer. Soc. Microb., #D-178, p.101) and Eikenella corrodens (Williams, L. H. et al (1990) Ann. Mtg., Amer. Soc. Microb., #D-61 p.90). They have even been observed in dental plaque by electron microscopy (Brady, J. M. et al (1977) J. Dent. Res 56:991-993; Halhoul, N. et al (1975) Arch. Oral Biol. 20:833-836). In spite of these reports, surprisingly little basic research has been done on oral phages, in view of their potential to affect bacterial populations in the oral cavity.
With regard to their function in dental plaque, phages are likely to influence the plaque flora in several potentially significant ways. Prophages, for example, provide immunity to super-infection by homoimmune phages and would presumably assist lysogens which carry them in competing with other bacteria in plaque by killing phage-sensitive competitors in a manner analogous to bacteriocinogenic cells. The semi-solid nature of dental plaque provides an especially favorable environment for this type of competition. Alternatively, lytic phage would be expected to select for phage-resistant mutants of sensitive strains and for mucoid mutants (phenotypically phage-resistant), which could well have altered colonizing and pathogenic properties. Actinophage-resistant mutants have in fact already been used to study cell surface structures that appear to be involved in specific, intergeneric oral bacterial coagreggation reactions (Delisle, A. L. et al (1988) Infect. Immun. 56:54-59; Tylenda, C. A. et al (1985) Infect. Immun. 48:228-233), which are believed to play an important role in colonization of dental plaque (Kolenbrander, P. E. et al (1985) In, S. E. Murgenhagen and B. Rosan (eds) pp. 164-171, American Society for Microbiology, Washington, D.C.).
The literature on S. mutans phages dates back to 1970, when Greer first claimed to be able to induce phages, by treatment with mitomycin C, from oral streptococcal strains AHT, BHT and HHT (Greer, S. W., et al (1970) IADR Abstr. 160; J. Dent. Res, 48A:88) and subsequently claimed that the same virus was present in all of eight cariogenic streptococci he examined, but not in non-cariogenic strains (Greer, S. W., et al (1971) J. Dent. Res. 50:1594-1604). He then reported that lysogens could be cured of their prophages by treatment with acridine orange (Greer, S. W., et al (1971) IADR Abstr. 57: J. Dent. Res. 49:67) and nitrosoguanidine (Greer, S. W., et al (1972) IADR Abstr. 68: J. Dent. Res. 50:65). The latter was used to isolate temperature-sensitive mutants, one of which was heat-inducible and could be used to obtain cured cells by brief heating. Greer also proposed a curing procedure based on radiosensitization of DNA by incorporating 5-bromodeoxyuridine lysogens (Ramberg, E. et al (1973) IADR Abstr. 113: J. Dent. Res. 52a), but its application to S. mutans was never subsequently reported. Greer never reported the successful isolation of an infectious phage which could be grown in S. mutans. Difficulties in repeating Greer""s induction experiments led many microbiologists to assume that he was really working with enterococci, which were common contaminants in the oral streptococcal cultures being exchanged among various laboratories during this time.
Feary was the next to report isolating phages for S. mutans (Feary, T. W. (1972) IADR Abstr. 67: J. Dent. Res. 50:65), from sewage, but all of his phage-sensitive strains were group D enterococci.
Klein and Frank also reported the presence of phages in cariogenic streptococci (and Actinomyces) (Klein et al (1973) J. Biol. Buccale 1:79-85), and later claimed that cured strains (isolated as survivors of heavy UV irradiation or treatment with acriflavine) of S. mutans OMZ 61 and 71 produced less extracellular insoluble polysaccharides from sucrose but were more cariogenic than their parent strains (Klein, J. P., et al (1975) J. Biol. Buccale 3:65-75). Unfortunately, their cultures were not studied by others and their results have not been confirmed. Like Greer, they did not isolate infectious phages which could grow in S. mutans. 
Higuchi et al ((1977) Infect. Immun. 15:938-944) induced a phage out of xe2x80x9cmucoidxe2x80x9d S. mutans strain PK1 with mitomycin C and claimed that by using it to infect (or transfect) a rough, non-adherent mutant of this strain (which they believed to be a cured derivative) they could obtain transductants (and transfectants) that were mucoid, very adherent and contained the phage but which were also converted to an arg+ phenotype. The latter observation, since the parent strain was argxe2x88x92, suggests that the transductants were really strains of S. sanguis; also their lysogenic culture, PK1, is an unusual strain which most workers now believe is not S. mutans. These authors reported that transfection of S. sanguis 10556 with phage PK1 DNA yielded mucoid, adherent mutants which produced large amounts of levan (Higuchi, M. et al (1977) Infect. Immun. 15:945-949). These colonies have the appearance of typical S. salivarius colonies; also, since they were argxe2x88x92 (which is characteristic of S. salivarius), whereas the parent strain (10556) was arg+, the validity of the results is very questionable.
Upon reviewing the literature on S. mutans phages, the existence of such phages has not been confirmed, except in the case of lytic phages isolated by Armau (Armau et al (1988) Ann. Mtg. IADR, J. Dent. Res. 67:121, Abstr. No. 69). Armau isolated 23 S. mutans phages from 3,974 dental plaque samples, using 17 test host strains. Nine were isolated on three serotype c strains, nine on one serotype e strain, one on the serotype f strain and four on two serotype d strains. No phages were isolated for one serotype b strain or two serotype g strains tested. All of the phages proved to serotype-specific. Four of the phages plated with reduced efficiency on different strains of the same serotype, suggesting the presence of restriction/modification systems.
Prior art methods for combatting the oral bacteria which lead to dental caries have relied on physical or chemical treatments to remove plaque or kill microorganisms, in a non-specific manner. Desirable organisms were therefore removed along with the target organisms. In the case of antibiotic treatments, resistant mutants often developed, rendering further treatment ineffective.
Current treatments which claim to reduce the numbers of organisms in dental plaque include a number of mouthwashes (rinses) that contain a variety of bacteriostatic and bacteriocidal organic chemicals. These chemicals include phenols, alcohols, peroxides, detergents/surfactants, quaternary ammonium compounds, root extracts (sanguinarine) and fluorides. A mouthrinse containing the bis-biguanide antibiotic chlorhexidine is now available, by prescription only, in the U.S. With the exception of fluorides and chlorhexidine, none of the currently available oral health care products have been demonstrated to be highly therapeutically effective in reducing plaque or preventing caries.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of treating and preventing dental caries and periodontal diseases, it should be apparent that there still exists a need in the art for a method of effectively combatting the oral bacteria which lead to these conditions.
In contrast to prior art methods of treating and preventing dental caries and periodontal diseases, the phage-encoded enzymes of the present invention do not lead to development of resistant bacterial mutants, because their development in response to the present compositions would require too drastic an alteration in the basic structure of the bacterial cell wall.
Accordingly, a major object of the present invention is to provide a method for the treatment and prevention of dental caries and periodontal diseases using phage-encoded anti-bacterial enzymes to kill the organisms causing these diseases in the oral cavity.
Another object of the present invention is to provide a method for studying the cell wall of an oral bacterium by treating the bacterium with a phage-encoded enzyme which degrades the cell wall.
A further object of the present invention is to provide a method for preventing spoilage of fresh, refrigerated or pasteurized perishable items by treating these items with a phage-encoded anti-bacterial enzyme which inhibits growth of a gram-positive spoilage bacterium on the items.
Another object is to provide a method for the removal of insoluble dextran polysaccharides by treating with a phage-encoded enzyme.
A still further object of the present invention is to provide a method for the removal of dental plaque by treating the oral cavity with a phage-encoded enzyme.
A still further object of the present invention is to provide an isolated and purified phage-encoded anti-bacterial enzyme which inhibits the establishment of an oral bacterium.
Another object of the present invention is to provide an isolated and purified phage-encoded enzyme which can be used to remove insoluble dextran polysaccharides.
Yet another object of the present invention is to provide an isolated and purified phage-encoded enzyme which can be used to remove dental plaque.
Another object of the present invention is to provide DNA fragments isolated from bacteriophage which encode the enzymes of the present invention.
A further object of the present invention is to provide an expression vector containing the DNA fragments encoding the enzymes of the present invention.
A still further object of the present invention is to provide a host cell containing an expression vector including DNA fragments encoding the enzymes of the present invention.
Another object of the present invention is to provide antibodies to the enzymes of the present invention.
A still further object of the present invention is to provide vehicles for supplying and treating with the enzymes of the present invention.
Yet another object of the present invention is to provide a genetically engineered non-cariogenic organism which produces phage-encoded enzymes that inhibit the establishment of a cariogenic organism.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.